Antiretroviral therapy has become a reality in resource-limited settings thanks to entities such as PEFAR and the Global Fund. However, contrary to the therapeutic choice flexibility in developed countries, all patients go on reverse transcriptase inhibitor (RTI)- based first-line regimens with more expensive protease and integrase inhibitor-based regimens being reserved as second-line therapy for those who fail initial treatment. RTI efficacy is compromised to a large degree by resistance. Because of cost, however, resistance testing is not performed and a high percentage of individuals fail treatment because of pre-existing resistance. This has undermined the efficacy of treatment rollout and has created an urgent clinical need. The solution is implementation of a simple, inexpensive assay for detection of resistance to first-line RTI-based regimens. We have exploited a novel polymerase, with extraordinary requirement for base pairing at the 3 end of the primer/target template, to create an allele specific (AS)-PCR assay that uses standard PCR and simply scores for resistance by the presence of amplification products. This format brings the cost of resistance testing down to one- tenth of the cost of current, gold-standard genotyping assays and would make it feasible, for the first time, to initiate wide-spread resistance testing in resource-limited settings. The proposed research will have 3 specific aims: 1) Aim 1: Design and optimize the AS-PCR assay to detect the six mutations that at clinically relevant frequencies confer resistance to first line antiretroviral therapy commonly used in resource-limited settings and assess the performance of the assay after modifying the primers by adding detection markers. 2) Aim 2: Determine the limits of multiplexing to minimize the number of reactions and reduce assay cost. 3) Aim 3: Assess the discriminatory capacity of the assay on validated HIV-1 sample panels with multiple clades and determine level of concordance with patient samples previously characterized by commercial genotyping assays. Completion of these aims will lead to a phase II submission focused on optimization of the assay for utilization in resource limited settings.